Copper transport from ceruloplasmin: characterization of the cellular uptake mechanism.
Copper uptake from 67Cu-labeled ceruloplasmin (67CuCp) was studied in K-562 cells, a human erythroleukemic cell line. 67CuCp was prepared by an ascorbate-catalyzed exchange of recrystallized ceruloplasmin with 67CuCl2. The labeled protein was treated with Chelex-100 and gel filtration to ensure that 67Cu was tightly bound to the structure. 67CuCp bound specifically to the K-562 cells at 4 degrees C. The binding was linear with protein in the range of 200-800 nM and in the presence of 3% albumin. In this concentration range, 67CuCl2 showed no binding that could be interpreted as specific; 80-90% of the cell-bound 67Cu was removed by washing the cells with acid buffer. When binding was attempted at 37 degrees C, a significant fraction of the 67Cu resisted acid washing and with time accumulated in the cells. Fractionating the cytosolic components on Percoll gradients located the 67Cu in buoyant fractions of densities 1.030-1.05, with a peak at 1.035. Repeating the experiment with 125I-labeled ceruloplasmin failed to localize any 125I label in Percoll fractions; very little 125I was detected in the cytosol. Double-labeled 67Cu-125I-ceruloplasmin confirmed that copper and not the protein moiety of ceruloplasmin was taken up by the cells. The uptake reaction was inhibited by 1 mM bathocuproine sulfonate and by 1 mM sodium iproniazid. Ascorbate (100 microM) strongly stimulated uptake. These studies provide evidence that K-562 cells are able to extract copper atoms from ceruloplasmin and transport the copper to the cytosol.(ABSTRACT TRUNCATED AT 250 WORDS)
Percival
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Department of Biochemistry and Biophysics, Texas A & M University, College Station 77843.
Harris
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D001692:Biological Transport; D002458:Cell Fractionation; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002570:Ceruloplasmin / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D011485:Protein Binding; D013347:Subcellular Fractions / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000378:metabolism
D003300:Copper; D002570:Ceruloplasmin